Discovery and Molecular Elucidation of the Anti-Influenza Material Basis of Banlangen Granules Based on Biological Activities and Ultra-High Performance Liquid Chromatography Coupled with Quadrupole-Orbitrap Mass Spectrometry

Abstract

Ethnopharmacological relevance: Traditional Chinese medicine (TCM) has a wide range of applications, including bioactive compound discovery and other human healthcare-associated applications. However, complex chemical systems present a significant challenge for chemical-material-based research and quality control. For instance, Banlangen (BLG) granules are a common TCM preparation with an anti-influenza effect which is widely used in clinical treatment, however, the chemical basis of their anti-influenza activity remains unclear.

Aim of the study: In the present study, a systematic discovery strategy for identifying anti-influenza molecules based on biological activities and chemical analysis was established to contribute to the molecular elucidation of the anti-influenza material basis of Banlangen granules.

Materials and Methods: Hemagglutinase inhibition (HAI) and neuraminidase inhibition (NAI) assays of H1N1, H5N1 and H7N9 were used to compare the anti-influenza activities of different fractions of BLG granules. A comparative qualitative analysis of the chemical constituents in BLG granules and their fractions was performed using ultra-high-performance liquid chromatography coupled with quadrupole orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap MS), in which a multiple mass spectrometry database platform and three compound identification strategies were used. The association between anti-influenza activities and chemical constituent characteristics was analyzed using multiple stoichiometries and data comparison strategies.

Results: The results showed that the chromatography fractions F3 and F4 of the BLG granules showed the highest anti-influenza activity. A total of 88 compounds were identified in the BLG granules, including 31 alkaloids, 16 organic acids, 10 nucleosides, eight phenylpropanoids, six sulfur-containing compounds, five amino acids, four aromatic compounds, three aldehydes and ketones, two flavonoids, one alcohol, one carbohydrate, and one aliphatic compound. Out of these, 6-methoxyquinoline and 4-guanidinobutanal were identified in BLG granules and their herbal-Isatidis Radix for the first time. Additionally, 31 characteristic compounds were identified in fractions F3–F4 as potential compounds with anti-influenza activity.

Conclusion: In this study, we proposed a systematic discovery strategy to thoroughly investigate the anti-influenza activity, chemical identification, and constituents-activity relationship of BLG granules. These data provide a deeper understanding of the molecular mechanism of the activity of BLG granules. Moreover, we present a basis for the discovery of potential novel drug candidates, elucidation of the molecular anti-influenza mechanisms, and quality evaluation and control of BLG granules.

Keywords: Banlangen granules, anti-influenza activity, UHPLC-Q-Exactive Orbitrap MS, chemical constituents, Traditional Chinese medicine